Advancing Asthma Drug Discovery and Translational Research with a Human Air-Liquid Interface (ALI) Asthma Model

A donor-derived airway epithelial model designed to capture key features of Type 2 asthma and support translational drug discovery.

Asthma remains one of the most common chronic respiratory diseases worldwide, affecting an estimated 363 million people and imposing a substantial burden on patients and healthcare systems worldwide [1]. Despite significant advances in biologic therapies, developing new treatments remains challenging due to the complex and heterogeneous nature of the disease. Asthma encompasses multiple inflammatory endotypes, including allergic and eosinophilic Type 2 (Th2) disease, neutrophilic asthma and paucigranulocytic asthma, each characterised by distinct underlying cellular and molecular mechanisms [2]. 

Regulatory agencies including the Food Drug and Administration (FDA) are increasingly supporting the development and adoption of New Approach Methodologies (NAMs), which aim to provide more human-relevant data while reducing dependence on animal studies [3]. Human donor-derived air-liquid interface (ALI) models represent one example of how respiratory drug developers can generate mechanistic and translational insights earlier in discovery. Here at RoukenBio, we are at the forefront of supporting drug discovery and therefore see the importance of early human-relevant models in the drug discovery pipeline.

For drug developers, selecting experimental models that accurately recapitulate key features of human respiratory disease remains a significant challenge. Conventional submerged cell culture systems, where airway epithelial cells are cultured as two-dimensional monolayers fully immersed in growth medium, often fail to reflect the airway epithelium's full structural and functional complexity [4]. Similarly, commonly used animal models, such as ovalbumin- or house dust mite-induced mouse models of asthma, may not fully mirror human-specific physiology, immune responses or disease mechanisms. As a result, there is growing demand for human-relevant experimental systems that bridge the translational gap between simplified in vitro assays and more advanced stages of preclinical and clinical development. 

The advantages of a Human Air-Liquid Interface (ALI) Asthma Model compared to other models

"One of the major challenges in respiratory drug discovery is identifying experimental systems that capture clinically relevant epithelial biology while remaining scalable for drug screening." - Dr Athanasios Koulis, KTP Associate at RoukenBio

To address this need, RoukenBio has developed a donor-derived air-liquid interface (ALI) lung airway model, with its first application focused on capturing key epithelial features of Type 2 airway inflammation and generating quantifiable endpoints to support therapeutic evaluation.

Why model the airway at an Air-Liquid Interface (ALI)?

The airway epithelium is a highly polarised barrier composed of specialised cell types, including ciliated, goblet and basal cells, whose coordinated functions regulate mucociliary clearance, maintain airway homeostasis and orchestrate responses to allergens, pathogens and inflammatory stimuli. In patients with Type 2 asthma, the secretion of cytokines such as interleukin-4 (IL-4) and interleukin-13 (IL-13) results in mucus hypersecretion, altered epithelial differentiation and airway remodelling [2, 5].

The ALI approach aims to recreate important features of the human airway microenvironment. In this system, primary human bronchial epithelial cells (HBECs) are cultured on permeable inserts with culture medium supplied to the basal surface while the apical surface remains exposed to air. Over a differentiation period of approximately three to four weeks, cells develop into a polarised three-dimensional pseudostratified mucociliary epithelium containing specialised airway cell populations, including mucus-secreting goblet cells and ciliated epithelial cells. 

This architecture more closely represents the organisation of the human airway than conventional submerged cultures and provides an opportunity to investigate epithelial responses in a biologically meaningful setting.

Establishing a Human ALI Model for Asthma Therapeutics

A key focus of RoukenBio's Innovation programme has been to generate a reproducible Type 2 asthma-like phenotype within differentiated ALI cultures.

Initial characterisation demonstrated successful epithelial differentiation, with expression of MUC5AC confirming goblet-cell development and β-tubulin staining demonstrating the presence of cilia. Together, these markers indicate the formation of a mature mucociliary epithelium, a critical pre-requisite for modelling airway disease.

To induce asthma-associated biology, differentiated cultures were exposed to IL-13 and related Type 2 cytokine stimuli. Histological analysis revealed substantial structural changes consistent with airway remodelling observed in patients. Compared with unstimulated cultures, IL-13-treated epithelia displayed increased thickness, altered organisation and accumulation of extracellular material and mucus. These observations were supported by confocal imaging, which demonstrated marked changes in epithelial architecture and elevated MUC5AC staining patterns following cytokine stimulation. 

"By combining donor-derived cells and clinically relevant biomarkers, we aim to generate data that better reflects human respiratory disease biology." - Dr Daria Paruzina, Director of Innovation at RoukenBio

A further hallmark of Type 2 asthma is excessive mucus production. Within the ALI system, IL-13 stimulation increased visible mucus formation, creating a quantifiable imaging endpoint readout that can be used to assess pharmacological activity. This provides a direct method for evaluating effect of a therapeutic compound on one of the most clinically relevant features of airway disease.

Increasing IL-13 stimulation drives quantifiable mucus formation in ALI lung cultures. Data shown from day 23 cultures. Internal data produced by RoukenBio.

In parallel, RoukenBio assessed soluble biomarkers associated with Type 2 inflammation. Notably, IL-13 stimulation produced concentration-dependent increases in CCL26 (eotaxin-3), a chemokine strongly linked to eosinophilic asthma biology and expression of periostin which is correlated with the thickness of airway basement membrane in asthma patients [6]. Further assay readouts including MUC5AC quantification in apical washes of the epithelium are being developed to expand the translational utility of the platform.
 

Increasing IL-13 stimulation drives dose dependent increase of CCL26 production across time. ELISA performed on basal culture supernatants. Internal data produced by RoukenBio.

Demonstrating Pharmacological Responsiveness in Asthma Drug Discovery

For any disease model, demonstrating responsiveness to pathway-specific intervention is an important step in establishing utility.

In a first proof-of-concept, treatment with dupilumab, an IL-4Rα-blocking monoclonal antibody that inhibits IL-4 and IL-13 signalling [7], reduced mucus-droplet formation in the RoukenBio donor-derived organotypic in vitro ALI cultures stimulated with IL-4, IL-13 or a combination of both cytokines. These findings support the underlying role of IL-4 and IL-13 signalling in driving the observed phenotype and demonstrate that the model can detect biologically meaningful responses to therapeutic intervention.

Addition of dupilumab (150 ng/ml) reduces mucus droplet area in IL-4 -/+ IL-13 stimulated ALI cultures. Data shown from day 21 cultures. Internal data produced by RoukenBio.

While no in vitro system can fully replicate the complexity of human asthma, these observations indicate that the platform captures several of the key epithelial features associated with Type 2 airway inflammation and can provide a useful tool for translational research and compound profiling. 

What Does This Mean for Drug Developers Working on Asthma Therapeutics?

RoukenBio's R&D investment in this model is driven by the increasing demand for human-relevant systems that can support target validation, mechanism-of-action studies and therapeutic evaluation.

Potential applications include:

• Assessment of IL-4 and IL-13 pathway inhibitors, including biologics and small molecules.
• Evaluation effects of compounds on mucus production and airway epithelial remodelling.
• Comparative profiling of healthy and disease-derived donor cells.
• Investigation of donor-to-donor variability and disease heterogeneity.
• Measurement of imaging, histological and soluble biomarker endpoints within a single experimental platform.
• Generation of translational datasets to support compound prioritisation and programme progression.

By combining donor-derived biology with multiple quantifiable readouts, the platform is designed to help drug developers understand whether their candidate therapies engage relevant airway epithelial mechanisms early in development.

Building Towards a High-Content Assay System with a Donor-Derived Organotypic In Vitro ALI Asthma Model

The current ALI platform provides a strong foundation for future expansion. RoukenBio is now progressing the donor-derived organotypic ALI asthma model towards a miniaturised 96-well transwell format intended to enable broader donor profiling and higher-throughput compound evaluation.

Ongoing development efforts include expanding donor-cell biobanks, optimisation of miniaturised culture conditions, refinement of imaging workflows and evaluation of pharmacological interventions across both healthy and asthma-derived donors. The long-term objective is to establish a scalable high-content screening platform capable of supporting asthma and other Type 2 inflammation-focused drug discovery programmes. 

Additional programme milestones include modelling other asthma subtypes and the introduction of different cell types critical in asthma pathogenesis to create the next generation of complex organotypic ALI asthma models.

Looking Ahead to Asthma Drug Discovery

As the pharmaceutical industry continues to seek more predictive and translatable preclinical models, human-relevant in vitro systems are becoming increasingly important in respiratory drug discovery. By integrating differentiated airway epithelial cultures, disease-relevant cytokine stimulation and a range of quantitative functional and molecular endpoints, RoukenBio's donor-derived ALI asthma model provides a physiological relevant platform for investigating Type 2 airway inflammation. 

Designed to support mechanism-of-action studies, biomarker discovery, and therapeutic profiling, this advanced human airway model enables researchers to generate deeper biological insights and assess candidate therapies in a setting that more closely reflects human disease. As the field moves towards more predictive and clinically relevant experimental systems, donor-derived ALI models represent an important step in bridging the gap between conventional cell-based assays and patient outcomes.

Interested in exploring how this ALI asthma model could support your respiratory, immunology or biologics programme? Contact RoukenBio to discuss how the ALI platform can be configured around your therapeutic target, modality and research objectives.

References

1. World Health Organization. Asthma Fact Sheet. 2026.
2. Gonzalez-Uribe C. et al. Human airway epithelial models in asthma research. 2023.
3. FDA Achieves Year 1 Goals in Reducing Animal Testing in Drug Development. New Approach Methodologies (NAMs) | FDA
4. Hiemstra P. et al. Airway and alveolar epithelial cells in culture. European Respiratory Journal. 2019.
5. Kraft M. Asthma Phenotypes and Interleukin-13 - Moving Closer to Personalized Medicine. N Engl J Med. 2015.
6. Matsumoto H. (2019). Roles of Periostin in Asthma. In: Kudo, A. (eds) Periostin. Advances in Experimental Medicine and Biology, vol 1132. Springer, Singapore. 2019.
7. Ferrante G. et al Severe pediatric asthma therapy: Dupilumab. Front. Pediatr. 10:963610. 2022.